Prodrug using nitroimidazole

ABSTRACT

Provided is a prodrug of 2-nitro-1-imidazolepropionic acid and a therapeutically active organic compound having on the molecule an amino group, a cyclic amino group or a hydroxyl group, particularly a prodrug in which the therapeutically active organic compound is selected from among antitumor agents. The prodrug cleaves specifically under hypoxic conditions in vivo to exhibit the inherent therapeutic activity.

TECHNICAL FIELD

This invention relates to a conjugate of 2-nitro-1-imidazolepropionicacid and a drug that is a therapeutically active compound. Morespecifically, the invention relates to such a prodrug in which acovalent bond between nitroimidazole and the drug cleaves at a hypoxicregion in a living organism or in a reducing environment, enabling atherapeutically active compound to be released in an active form, and tothe use of 2-nitro-1-imidazolepropionic acid for providing such aconjugate.

BACKGROUND ART

Tumors at hypoxic regions cause invasive, metastatic and chemoresistantcancers, and are the biggest factor preventing a complete cure ofcancer. There is an urgent desire for the development of methods fortreating tumor cells in such low-oxygen environments.

Several kinds of clinical trials are currently being conducted on thefollowing and other drugs targeted at cancer cells in hypoxic regions:triapazamine, AQ4N (banoxantrone dihydrochloride), PR104(dinitrobenzamide nitrogen mustard prodrug) and TH-302(N,N′-bis(2-bromoethyl)phosphorodiamidic acid(1-methyl-2-nitro-1H-imidazol-5-yl) methyl ester. However, at present,there is no information to indicate that these have been brought tomarket.

Dinitrobenzamide and other compounds which act as such protecting groupsare disclosed in Non-Patent Document 1 and nitroimidazoles relating toTH-302 are disclosed in Non-Patent Document 2, Patent Document 1 andPatent Document 2. TH-302, as shown in the following reaction scheme, isunderstood to utilize the mechanism of cleaving the protectinggroup-drug bond to release the drug.

In both Patent Documents 1 and 2, the drug is bonded through amethyleneoxy group (—CH₂—O—) to a carbon atom on the imidazole ring, andis understood to be released from the prodrug by the same mechanism asin the above reaction scheme. However, in this system, the efficiency ofdrug release is dependent on the leaving ability of the drug; the drugthat is released is limited to a compound having a phenolic hydroxylgroup or phosphoric acid that readily dissociates.

Patent Document 3 mentions a hypoxia-activated conjugate (Hyp-L-Q)obtained by covalently bonding a bioreductive group (Hyp: e.g.,2-nitroimidazolyl) at the 1 position thereon through a linker (L:—CH₂CH₂CH₂—C═O)—) to the amino group on an anthracycline-basedanticancer agent (Q). With regard to this compound which is a conjugate,in a hypoxic tumor region, although the -L-Q moiety remained bonded, thenitro group on 2-nitroimidazolyl is reduced to hydroxyamine, whichsuggests that DNA can be alkylated through the 4- or 5-position onimidazolyl and that, at the same time, the anthracycline making up Q isintercalated between DNA bases, thereby killing cancer cells. However,it was revealed in this patent publication that a compound in which thelinker is “—C3H6-C(═O)—”, as compared with a corresponding compound inwhich the linker is —CH₂(CH₂)_(a)CH₂—O—CH₂— (“a” here being the integer0 or 1), for example, exhibits only a very low cytotoxicity to lungcancer cells under hypoxic conditions. Hence, such a compound fallsoutside the scope of the claims of this patent application.

In addition, Patent Document 4 mentions that amides such asN-methyl-2-nitro-1-imidazole propanoylamide, for example, have aradiation-sensitizing action and also have, by themselves, an anticancereffect. However, no mention or suggestion whatsoever has been made thatsuch imidazole carboxylic acids can be used to form conjugates orprodrugs with other drugs.

CITATION LIST Patent Literature Document

-   Patent Document 1: WO 2000/64864 or Japanese Translation of PCT    Application No. 2002-543059-   Patent Document 2: WO 2004/087075 or Japanese Translation of PCT    Application No. 2006-521409-   Patent Document 3: WO 2009/018163 A1-   Patent Document 4: JP Hei 7-101860 A (1995)

Non-Patent Literature Document

-   Non-Patent Document 1: B. M Sykes et al., J. Med. Chem. 42, 345-355    (1999)-   Non-Patent Document 2: J. Duan et al., J Med. Chem. 51(8): 2412-2420    (2008)

SUMMARY OF THE INVENTION Technical Problem

The inventors inferred that, because 2-nitroimidazole has a relativelyhigh redox potential and is efficiently reduced at hypoxic regions invivo, the nitro group is reduced to hydroxyamine and amine, and that,were it possible to apply the structural change in the molecule whichthus arises to the release of a compound, this would lead to thedevelopment of carcinostatic drugs specific to hypoxic regions. In otherwords, if a compound lacks activity at a normal oxygen concentrationbecause it exists in the form of a prodrug, but undergoes a structuralchange and assumes an active form in hypoxic regions, this should enablethe development of a pharmaceutical product which suppresses the sideeffects of the anticancer drug but is active in hypoxic regions.Accordingly, the object of this invention is to provide a system whichis more versatile than the conventional art and in which the molecularstructure specifically changes in hypoxic regions such as tumors,efficiently releasing a compound such as a drug.

Solution to Problem

It is implied in Non-Patent Document 1 that, in a compound having a drugcovalently bonded to the carbonyl group of the linker —CH(-Me)-C(═O)—which is itself bonded at the 1 position of a 2-nitroimidazole skeleton,an intramolecular cyclization reaction does not arise even when thenitroimidazole moiety is reduced, and so the drug is not released. Atthe same time, with regard to Hyp-L-Q in which the linker portion is—CH₂CH₂CH₂—C(═O)—, Patent Document 3 mentions that although the2-nitroimidazole moiety corresponding to Hyp is reduced under hypoxicconditions, the L-Q bond does not cleave.

Surprisingly, the inventors have found that an amide bond, imide bond orester bond that has been formed through such a carbonyl group cleaves ina reducing environment, especially at hypoxic regions in vivo.

Such cleavage, although not theoretically constrained, can be understoodas arising together with an intramolecular cyclization reactionaccording to the following reaction scheme in which the nitro group onthe imidazole ring is converted to a hydroxyamine or amino group in areducing environment, which amino group carries out intramolecularnucleophilic attack.

According to the present invention, there can be provided a compoundcapable of serving as a conjugate or prodrug for a wide variety ofdrugs, which compound, because the amide bond, imide bond or ester bondcleaves in a reducing environment, releasing a drug having its ownactivity at that place, is technically meaningful in that release occursselectively in a reducing environment.

Hence, this invention is based on the discovery that2-nitro-1-imidazolepropionic acid can be used to provide a wide varietyof drugs, especially prodrugs of therapeutically active organiccompounds.

In one aspect, the invention provides a compound represented by generalformula (I), or a pharmaceutically acceptable salt thereof,

wherein Z is of formula (a):

or formula (b)—O—R3  (b).In these formulas, R1 is a residue of an amino group-bearingtherapeutically active organic compound after removal of the amino grouptherefrom and R2 is a hydrogen atom, or R1 and R2 represent, togetherwith the adjoining nitrogen atom, a residue of a therapeutically activeorganic compound having a cyclic amino group; and R3 is a residue of ahydroxyl group-bearing therapeutically active organic compound afterremoval of the hydroxyl group therefrom.

In other aspects, the invention provides a preparation for producing aprodrug of a therapeutically active organic compound which has an aminogroup, a cyclic amino group or a hydroxyl group on the molecule andincludes 2-nitro-1-imidazolepropionic acid of formula (II)

as a reactant; and also provides the use of a compound of formula (II)as a reactant for producing a prodrug of a therapeutically activeorganic compound having on the molecule an amino group, a cyclic aminogroup or a hydroxyl group.

In such a compound represented by general formula (I), because2-nitro-1-imidazolepropionyl (sometimes abbreviated below as “Izp”) isbonded to a therapeutically active organic compound, it lowers or masksthe biological activity inherent to the organic compound (such ascytotoxicity and other activities), yet the Izp moiety and the moietycorresponding to Z selectively cleave in a reducing environment,particularly in hypoxic regions within mammals. The inherent activity ofthe organic compound furnished by such cleavage is exhibited at hypoxicregions and the periphery thereof.

Accordingly, the compound represented by general formula (I) is able torelease the therapeutically active organic compound included therein atspecific regions within a mammal, thereby enabling safer and moreeffective use.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, technical terms used in this specification orused with regard to this invention bear the meaning and import withwhich they are generally used in the technical field to which thisinvention pertains.

As used herein, “prodrug” has the meaning with which it is used withinthis technical field. For example, it refers to a compound whichchemically modifies a physiologically active substance or atherapeutically active organic compound and has been designed todissociate or release the parent compound in vivo under enzymatic orother conditions.

“Conjugate” refers to an entity formed by covalently bonding two or moredifferent compounds, and is used as a concept that encompasses prodrugs.

“Therapeutically active organic compound” refers to an organic compoundhaving a therapeutic or prophylactic activity against diseases,disorders or the like in mammals, particularly humans. Examples of suchdiseases and disorders include tumors, especially malignant tumors, andinflammation, which lesions or regions peripheral thereto are associatedwith a hypoxic state compared with normal tissue or cell regions.

“Antitumor drug or substance” and “anticancer drug” are used asinterchangeable terms.

Antitumor substances encompassed by therapeutically active organiccompounds include not only compounds currently used in cancerchemotherapy or being tested for use, but also compounds whose clinicaluse has been abandoned on account of strong toxicity or side effects,and compounds which will in the future be provided as anticancer drugs,so long as they suit the purposes of this invention. Such anticancerdrugs include, but are not limited to, anthracycline-based compoundssuch as doxorubicin, idarubicin, epirubicin, daunorubicin, pirarubicin,amrubicin, aclacinomycin, anthramycin and zorubicin; peptide-basedcompounds such as bleomycin and actinomycin; quinoline alkaloid-basedcompounds such as camptothecin, topotecan and irinotecan; taxane-basedcompounds such as docetaxel and paclitaxel; vinca alkaloid-basedcompounds such as vinorelbine, vincristine, vinblastine and vindesine;deoxycytidine-based compounds such as gemcitabine and cytarabine;pyrimidine-based compounds such as 5-fluorouracil, capecitabine anddoxyfluridine; purine ring derivative-based compounds such asfludarabine, 6-mercaptopurine and 6-thioguanine; macrolide-basedcompounds such as epothilone; amino acid derivative-based compounds suchas melphalan; and folic acid derivative-based compounds such asmethotrexate and pemetrexed.

Anti-inflammatory substances encompassed by therapeutically activeorganic compounds include salicylic acid-based non-steroidalanti-inflammatory drugs such as mesalazine; oxicam-type non-steroidalanti-inflammatory drugs such as piroxicam, meloxicam and tenoxicam; andsteroid-based anti-inflammatory drugs such as cortisone, hydrocortisone,cortisone acetate, prednisolone, methylprednisolone, betamethasone,dexamethasone, triamcinolone and triamcinolone acetonide.

Examples of the amino group, cyclic amino group or hydroxyl group whichis present within the molecule of these antitumor substances and can beused to form an amide bond, imide bond or ester bond by reaction withthe compound of formula (II) include the following: in anthracyclines,an amino group or hydroxyl group present on the sugar moiety; inpeptide-based antibiotics, an amino group; in quinoline alkaloids, ahydroxyl group on the E ring; in taxanes, a hydroxyl group bonded to thetaxane ring or a side-chain hydroxyl; in vinca alkaloids, a cyclic aminogroup on the indole ring; in deoxycytidine derivatives, an exocyclicamino group on the cytosine base or a hydroxyl on the ribose ring; inpyrimidine derivatives, a cyclic amino group on the pyrimidine ring or ahydroxyl on the ribose ring; in purine ring derivatives, a cyclic aminogroup or exocyclic amino group on the purine ring or a hydroxyl group onthe ribose ring; in macrolide antibiotics, a hydroxyl group on themacrolide ring; in amino acid derivatives, an amino group bonded to thea carbon; and in folic acid metabolic antagonists, an amino group bondedto the heterocycle.

Examples of the amino group, cyclic amino group or hydroxyl group whichis present on the molecule of an anti-inflammatory agent and may be usedto form an amide bond, imide bond or ester bond by reacting with theformula (II) compound include the following: in salicylic acid-basedcompounds, a hydroxyl group or amino group bonded to the benzene ring;in oxicam-based compounds, a hydroxyl group present on the cyclicsulfonamide; and in steroidal compounds, a hydroxyl group bonded to thecarbon at position 21.

Therefore, illustrative examples of compounds of general formula (I)which are amino group-bearing therapeutically active organic compoundsinclude doxorubicin, idarubicin, epirubicin, daunorubicin, pirarubicin,amrubicin, aclacinomycin, anthramycin, zorubicin, bleomycin,actinomycin, gemcitabine, cytarabine, methotrexate, pemetrexed,melphalan and mesalazine;

which are cyclic amino group-bearing therapeutically active organiccompounds include vincristine, vinblastine, vindesine, 5-fluorouraciland 6-mercaptopurine; and

which are hydroxyl group-bearing therapeutically active organiccompounds include docetaxel, paclitaxel, vincristine, vinblastine,vindesine, doxorubicin, idarubicin, epirubicin, daunorubicin,pirarubicin, amrubicin, aclacinomycin, anthramycin, zorubicin,bleomycin, actinomycin, gemcitabine, cytarabine, capecitabine,doxyfluridine, epothilone, piroxicam, meloxicam, tenoxicam, cortisone,hydrocortisone, cortisone acetate, prednisolone, methylprednisolone,betamethasone, dexamethasone, triamcinolone and triamcinolone acetonide.

Of these, examples of preferred therapeutically active organic compoundsinclude doxorubicin, idarubicin, epirubicin, daunorubicin, pirarubicin,amrubicin, aclacinomycin, anthramycin, zorubicin, bleomycin,actinomycin, camptothecin, topotecan, irinotecan, docetaxel, paclitaxel,vinorelbine, vincristine, vinblastine, vindesine, gemcitabine,cytarabine, 5-fluorouracil, capecitabine, doxyfluridine, fludarabine,6-mercaptopurine, 6-thioguanine, epothilone, piroxicam, melphalan,methotrexate, pemetrexed, mesalazine, prednisolone, methylprednisolone,dexamethasone and betamethasone. Especially preferred examples includedoxorubicin, idarubicin, epirubicin, daunorubicin, pirarubicin,amrubicin, aclacinomycin, anthramycin, zorubicin, gemcitabine,cytarabine, methotrexate, pemetrexed, melphalan, mesalazine,vincristine, vinblastine, vindesine, 5-fluorouracil, 6-mercaptopurineand prednisolone.

Pharmaceutically acceptable salts of the compound represented by generalformula (I), in cases where the compound has a basic group other thanthe amino group or cyclic amino group that forms an amide bond or imidebond, may be acid addition salts of mineral acids such as hydrochloricacid and sulfuric acid or organic acids such as formic acid, aceticacid, citric acid and methanesulfonic acid. In cases where the compoundhas an acidic group such as a carboxyl group or a hydroxyl group,pharmaceutically acceptable salts of the compound may be base additionsalts of alkali metals such as sodium and potassium, ammonium, ororganic amines such as methylamine.

In this invention, “hypoxic region” is used without regard to whetherthe site is in vivo or in vitro, although this term encompasses inparticular solid cancer lesions or solid cancer cell masses, and regionsperipheral thereto, preferably in living mammals, and especially humans.

Production of the compound represented by general formula (I) by thereaction of a therapeutically active organic compound having an aminogroup, a cyclic amino group and/or a hydroxyl group on the molecule witha compound of formula (II) to form an amide bond, an imide bond or anester bond may be carried out by reacting the organic compound with acompound of formula (II) within a suitable inert solvent and in thepresence of a condensing agent (e.g., a carbodiimide) that is itselfcommonly known in the technical field pertaining to the invention, or byreacting an active ester (e.g., an ester with a halide orN-hydroxysuccinimide) of the compound of formula (II) with the compoundrepresented by general formula (I) in a suitable solvent. When an aminogroup or cyclic amino group and a hydroxyl group are both present on themolecule of the organic compound, if necessary, one of the abovereactions may be carried out after either group has been protected by amethod that is familiar within the field of the invention.

The compound or prodrug of general formula (I) may be administered tothe patient in the same dosage form and by the same route ofadministration as the therapeutically active organic compound serving asthe parent compound. Although not subject to any particular limitation,the pharmaceutical preparation may be prepared using a pharmaceuticallyacceptable carrier. For example, pharmaceutical preparations suitablefor parenteral or intramuscular administration may be prepared bydissolution or suspension in an aqueous or nonaqueous solution ordilution in which a buffer, a tonicity modifier and the like have beenincluded and which optionally includes, for example, a surfactant, aliposome-forming agent, a polymer micelle-forming agent and the like.The amount administered should be set while referring to the dose of theparent compound and, where necessary, consulting with a specialist.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an NMR chart of Compound 4 synthesized by a cyclizationreaction in Example 1.

FIG. 2 is a graph showing the results of high-performance liquidchromatography (HPLC) comparing the amount of naphthylmethylaminereleased in normal oxygen and low-oxygen environments in Example 4.

FIG. 3 is a graph showing the release behavior of an amino group-bearingorganic compound from a model compound represented by general formula(I) using human pancreatic cancer cells in Example 5.

FIG. 4 is a graph showing the results of an evaluation of cell survivalrates in Example 7 (hypoxic environment-responsive doxorubicin).

FIG. 5 is a graph showing the results of a comparative test on thecytotoxicity of known compounds that are structurally analogous to thecompounds of the invention in a comparative test example.

FIG. 6 is a graph showing the results of a cell survival rate evaluation(hypoxic environment-responsive gemcitabine) in Example 9.

FIG. 7 is a graph showing the results of a cell survival rate evaluation(hypoxic environment-responsive 5-fluorourasil) in Example 11.

FIG. 8 is a graph showing the drug release behavior from a prednisoloneprodrug within a hypoxic environment in Example 16.

DESCRIPTION OF THE EMBODIMENTS

The invention is described more concretely below by way of examples,although the examples are not intended to limit the invention.

Example 1 Reduction of Methyl 3-(2-Nitro-1H-Imidazolyl)propionate

A reaction tube to which had been added 200 mg of Compound 1 synthesizedaccording to a method described in the non-patent document (M. P. Hay etal., J. Med. Chem. 37, 381-391 (1994)) was charged with 10 mL ofmethanol and 150 mg of Pd/C, then filled with hydrogen gas. With thetube filled with hydrogen gas, stirring was continued for 24 hours.Following the reaction, thin-layer chromatography (TLC) (developingsolvent, ethyl acetate:hexane=1:1) was used to confirm that the reactionhad proceeded. Next, the Pd/C was removed by Celite filtration and, lastof all, the methanol was removed using an evaporator. As a result, aring structure was rapidly formed in solution during the reducingreaction, without isolation of the Intermediate 3, yielding Compound 4.FIG. 1 shows an NMR chart of the Compound 4 thus obtained.

It was confirmed from this chart that Compound 4 having a cyclicstructure formed due to the reduction of Compound (1). Hence, it isapparent that, together with an intramolecular cyclization reaction bythe compound analog of formula (I), the ester bond cleaves, releasingHOMe.

Example 2 Preparation ofN-Naphthylmethyl-3-(2-Nitro-1H-Imidazolyl)propionylamide

A 50 mL round-bottomed flask was charged with Compound 2 (100 mg)synthesized according to a method described in the non-patent document(M. P. Hay et al., J. Med. Chem. 37, 381-391 (1994)), following which1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (155.6 mg),N,N-dimethyl-4-aminopyridine (6.7 mg), methylene chloride (5.4 mL) and1-naphthylmethylamine (119.27 μL) were added to the reactor understirring with a stirrer and stirring was continued for two days. A 100mL separatory funnel was charged with 20 mL of ethyl acetate and 20 mLof saturated ammonium chloride, and the organic phase and aqueous phasewere separated. A saturated aqueous solution of sodium bicarbonate and asaturated aqueous solution of NaCl were added to the recovered ethylacetate phase and separation was carried out, thereby recovering theorganic phase. The organic phase was dried over anhydrous Na₂SO₄,following which the solvent was removed with an evaporator. The residuewas isolated and purified by silica gel column chromatography(developing solvent, ethyl acetate:hexane=1:1; 400 mL), and the solventwas removed with an evaporator, yielding the target Compound 5.

ESI-MS (M+H⁺): theoretical value, 325.130; measured value, 325.126

Example 3 Reduction of Compound 5

A stirring bar and Compound 5 (10 mg) were added to a 50 mLround-bottomed flask. While stirring with the stirrer, 10 mL of methanoland 50 mg of Pd/C were added to the reactor, and the reactor was filledwith hydrogen gas. With the reactor filled with hydrogen gas, stirringwas continued for 24 hours. Following the reaction, TLC (developingsolvent, ethyl acetate:hexane=1:1) was used to confirm that the reactionhad proceeded. Next, the Pd/C was removed by Celite filtration and, lastof all, the methanol was removed using an evaporator. Analysis byelectrospray ionization mass spectroscopy (ESI-MS) yielded resultsindicating the formation of Compound 4. This showed that, together withan intramolecular cyclization reaction, the amide bond cleaved,releasing naphthylmethylamine.

Compound 4: theoretical molecular weight, 138.067; measured value,138.063

Example 4 Incubation of Compound 5 in Cultured Cells Under Low Oxygen

This example was carried out to confirm that Compound 5 is reduced incells within a hypoxic environment, and that naphthylmethylamine isreleased by a subsequent intramolecular cyclization reaction.

A cell suspension prepared to a cell count of 1×10⁴ cells/mL wasdispensed into a 96-well plate, and the cells were cultured by 24 hoursof incubation at 37° C. After 24 hours, the synthesized Compound 5 wasadded to the cells in an amount of 1 mM. Following addition, the cellswere cultured for 6 hours each in a normal oxygen concentrationincubator (20% O₂) or at a low-oxygen work station (1% O₂). The culturemedium was then recovered, 50 μL of Trypsin/EDTA was added and 5 minutesof incubation was carried out, following which the cells were strippedaway and added to the medium that was earlier recovered. Next, therecovered sample was freeze-dried overnight, 200 μL of acetonitrile wasadded, and 30 minutes of ultrasonic cleaning was carried out. Inaddition, dead cells were precipitated by centrifugal separation (3,000rpm, 10 min), the supernatant was recovered, and the acetonitrile wasremoved with a centrifugal evaporator. Next, 200 μL of methanol (forLC/MS) was added to the Eppendorf tube used in centrifugation, the tubecontents were passed through a filter (0.2 μm), and LC/MS measurementand high-performance liquid chromatography (HPLC) measurement werecarried out. By carrying out this measurement, the amount of compoundreleased in a low-oxygen environment and the amount released in a normaloxygen environment were compared. The HPLC results are shown in FIG. 2.It is apparent from the diagram that much naphthylmethylamine isreleased in a low-oxygen environment.

Example 5 Incubation of Compound 5 in Cultured Cells Under Low Oxygen

Human pancreatic cancer cells (MIA PaCa-2, acquired from Riken CellBank) were prepared as a suspension having a cell count of 1×10⁴cells/mL), and dispensed into a 96-well plate. After 24 hours, Compound5 was added to a concentration of 10 μM and the cells were cultured, ina normal oxygen concentration incubator (20% O₂) or at a low-oxygen workstation (0.1% O₂). After a given time had elapsed, the culture mediumwas recovered, trypsin was added and the cells were recovered. Therecovered cells were disrupted by sonic treatment, the compound wasextracted with acetonitrile, and analysis by LC/MS was carried out underthe following conditions.

Column used: Lachrom Ultra C18 (particle size, 2 μm; 2 mm×50 mm) column

Measurement wavelength: 220 nm

Eluent A: 0.1% TFA-containing milliQ

Eluent B: acetonitrile

Flow rate: 0.2 mL/min

Gradient: 95:5 (Eluent A:Eluent B)→95:5 (5 minutes)→30:70 (15 minutes)

By carrying out this measurement, the amount of compound released in alow-oxygen environment and the amount of compound released in a normaloxygen environment were compared. The HPLC results are shown in FIG. 3.It is apparent from the diagram than much naphthylmethylamine isreleased in a low-oxygen environment.

Example 6 Production of Doxorubicin Prodrug

(1) Synthesis of Compound 6

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (80.8 mg)was added to Compound 2 (60 mg) synthesized in the same way as inExample 2, following which N-hydroxysuccinimide (48 mg) was added andthe reaction was carried out in N,N-dimethylformamide (1 mL) for 1 hourunder ice cooling, then for 3 hours at room temperature. Following thereaction, several drops of acetic acid were added dropwise under icecooling and the system was stirred for 30 minutes. Partitioning betweenethyl acetate and saturated saline was carried out, after which theorganic phase was concentrated by evaporation. Next, 2-propanol wasadded and heating was carried out, after which impurities were filteredoff and the filtrate was ice-cooled, yielding Compound 6 (50 mg). ESI-MS(M+H⁺); theoretical value, 283.068; measured value, 283.079

(2) Synthesis of Compound 7

Compound 6 (2.2 mg) was added, within a mixed solvent ofN,N-dimethylformamide (50 μL) and water (50 μL), to doxorubicin (3 mg),after which triethylamine (1.4 μL) was added and the reaction wascarried out for 24 hours at room temperature. Following the reaction,purification was carried out with a reversed phase HPLC column (GLSciences Inc.; Inertsil ODS-3, 20×50 mm; flow rate, 5 mL/min; developingsolution, methanol/water=60/40 (0 min) to 100/0 (20 min)).

ESI-MS (M+Na⁺): theoretical value, 733.1967; measured value, 733.2013

Compound 7 in the above reaction scheme was thereby obtained.

Example 7 Evaluation of Cell Survival Rate (Low-OxygenEnvironment-Responsive Doxorubicin)

Human pancreatic cancer cells (MIA PaCa-2, acquired from Riken CellBank) were dispensed at a density of 5,000 cells/well and cultured for24 hours in Dulbecco's modified Eagle medium (DMEM), following whichCompound 7 was added to the specified concentration and 6 hours ofculturing was carried out at a normal oxygen concentration (20%) or alow oxygen concentration (0.1%). After culturing, DMEM mediumreplacement was carried out, the compound was removed, and 48 hours ofculturing was carried out in a normal oxygen concentration incubator,following which the cell survival rate was analyzed by a WST assay. Theresults are shown in FIG. 4.

It is apparent from the diagram that Compound 7 significantly lowers thesurvival rate of human pancreatic cancer cells at a low-oxygenconcentration compared with a normal oxygen concentration.

Comparative Example Comparison of Cytotoxicities of Inventive Compoundand a Known Compound that is Structurally Similar

Compound 8 below, which is mentioned in WO 2009/018163 A1, was furnishedfor use. The cell survival rates were compared in tests conducted by themethod described in Example 7 on both Compound 8 and a compound withinthe scope of this invention (aside from the fact that the correspondinglinker was —CH₂CH₂C(═O)—, the manner of bonding between nitroimidazoleand the drug was the same; Compound 7 of Example 6). The results areshown in FIG. 5.

From the diagram, compared with known Compound 8, Compound 7 accordingto this invention shows a significantly higher cytotoxicity againsthuman pancreatic cancer cells.

Example 8 Synthesis of Low Oxygen Environment-Responsive GemcitabineProdrug

Trimethylchlorosilane (47 μL) was added to gemcitabine (22 mg) dissolvedin pyridine (1 mL), and stirring was carried out for 2 hours at 0° C.Next, Compound 6 dissolved in acetonitrile (1 mL) was added and stirredfor 12 hours at 45° C. The reaction was followed by the addition ofethanol (1 mL) and 30 minutes of stirring at 45° C., then the additionof water (1 mL) and 30 minutes of stirring at 45° C. The solvent wasremoved by evaporation, followed by purification with a reversed phaseHPLC column (GL Sciences Inc.; Inertsil ODS-3, 20×50 mm; flow rate, 5mL/min; developing solution, acetonitrile/water=20/80 (0 min) to 50/50(30 min)).

This gave purified Compound 9 in a yield of 20%.

ESI-MS [M+H]⁺: theoretical value, 431.11; measured value, 431.20

Example 9 Evaluation of Cell Survival Rate (Low-OxygenEnvironment-Responsive Gemcitabine)

Human pancreatic cancer cells (MIA PaCa-2, acquired from Riken CellBank) were dispensed at a density of 5,000 cells/well and cultured for24 hours in DMEM medium, following which Compound 9 was added to thespecified concentration and 1 hour of culturing was carried out at anormal oxygen concentration (20%) or a low oxygen concentration (0.1%).After culturing, DMEM medium replacement was carried out, Compound 9 wasremoved, and 48 hours of culturing was carried out in a normal oxygenconcentration incubator, following which the cell survival rate wasanalyzed by a WST assay. The results are shown in FIG. 6.

Example 10 Synthesis of Low Oxygen Environment-Responsive FluorouracilProdrug

Thionyl chloride (500 μL) was added to Compound 2 (30 mg) dissolved inmethylene chloride (1 mL), and the reaction was carried out at 60° C.for 2 hours. Following the reaction, the solvent was removed byevaporation. The product (Compound 10) was dissolved in methylenechloride (1 mL), 5-FU (21 mg) dissolved in pyridine (1 mL) was added,and the reaction was carried out at 0° C. for 30 minutes, then at roomtemperature for 12 hours. After the reaction, the solvent was removed byevaporation and purification was carried out with a reversed phase HPLCcolumn (GL Sciences Inc.; Inertsil ODS-3, 20×50 mm; flow rate, 5 mL/min;developing solution, acetonitrile/water=20/80 (0 min) to 50/50 (30min)), thereby giving Compounds 11 and 12 as a mixture. The yield was45%.

ESI-MS [M+H]⁻: theoretical value, 296.1; measured value, 296.1

Example 11 Evaluation of Cell Survival Rate (Low OxygenEnvironment-Responsive 5-Fluorouracil)

Human pancreatic cancer cells (MIA PaCa-2, acquired from Riken CellBank) were dispensed at a density of 5,000 cells/well and cultured for24 hours in DMEM medium, following which a mixture of Compounds 11 and12 was added to the specified concentration and 24 hours of culturingwas carried out at a normal oxygen concentration (20%) or a low oxygenconcentration (0.1%). After culturing, medium replacement was carriedout, the compounds were removed, and 48 hours of culturing was carriedout in a normal oxygen concentration incubator, following which the cellsurvival rate was analyzed by a WST assay. The results are shown in FIG.7.

Example 12 Synthesis of Low Oxygen Environment-Responsive MesalazineProdrug

Compound 10 (0.11 mmol) synthesized in the same way as in Example 10 wasdissolved in methylene chloride (1 mL), then mesalazine (16 mg)dissolved in pyridine (1 mL) was added and the reaction was carried outat 0° C. for 30 minutes, then at room temperature for 24 hours. Afterthe reaction, the solvent was removed by evaporation, and purificationwas carried out with a reversed phase HPLC column (GL Sciences Inc.;Inertsil ODS-3, 20×50 mm; flow rate, 5 mL/min; developing solution,methanol/water=20/80 (0 min) to 20/80 (30 min)), thereby giving Compound14. The yield was 35%.

ESI-MS [M−H]⁻: theoretical value, 319.07; measured value, 318.78

Example 13 Synthesis of Low Oxygen Environment-Responsive MelphalanProdrug

Compound 10 (0.11 mmol) synthesized in the same way as in Example 10 wasdissolved in methylene chloride (1 mL), then melphalan (32 mg) dissolvedin pyridine (1 mL) was added and the reaction was carried out at 0° C.for 30 minutes, then at room temperature for 24 hours. After thereaction, the solvent was removed by evaporation and purification wascarried out with a reversed phase HPLC column (GL Sciences Inc.;Inertsil ODS-3, 20×50 mm; flow rate, 5 mL/min; developing solution,methanol/water=20/80 (0 min) to 80/20 (30 min)), thereby giving Compound15. The yield was 38%.

ESI-MS [M−H]⁻: theoretical value, 470.10; measured value, 469.63

Example 14 Synthesis of Low Oxygen Environment-Responsive MethotrexateProdrug

Compound 10 (0.11 mmol) synthesized in the same way as in Example 10 wasdissolved in methylene chloride (1 mL), then methotrexate (50 mg)dissolved in pyridine (1 mL) was added and the reaction was carried outat 0° C. for 30 minutes, then at room temperature for 24 hours. Afterthe reaction, the solvent was removed by evaporation, and purificationwas carried out with a reversed phase HPLC column (GL Sciences Inc.;Inertsil ODS-3, 20×50 mm; flow rate, 5 mL/min; developing solution,methanol/water=20/80 (0 min) to 80/20 (30 min)), thereby giving amixture of Compounds 16 and 17. The yield was 51%.

MALDI-TOF MS [M−H]⁻: theoretical value, 620.19; measured value, 620.38

Example 15 Synthesis of Low Oxygen Environment-Responsive PrednisoloneProdrug

Compound 10 (0.16 mmol) synthesized in the same way as in Example 10 wasdissolved in methylene chloride (1 mL), then prednisolone (84 mg)dissolved in pyridine (1 mL) was added and the reaction was carried outat 0° C. for 30 minutes, then at room temperature for 24 hours. Afterthe reaction, the solvent was removed by evaporation, partitioningbetween saturated saline and chloroform was carried out, and the organicphase was treated with sodium sulfate. The organic phase was thenconcentrated using an evaporator and purified by silica gelchromatography, giving Compound 13. The yield was 42%.

ESI-MS (M+H⁺): theoretical value, 528.23; measured value, 527.93

Example 16 Release of Drug from Prednisolone Prodrug in Low-OxygenEnvironment

Human pancreatic cancer cells (MIA PaCa-2, acquired from Riken CellBank) were prepared as a suspension having a cell count of 1×10⁴cells/mL), and dispensed into a 96-well plate. After 24 hours, Compound13 was added to a concentration of 10 and the cells were cultured in anormal oxygen concentration incubator (20% O₂) or at a low-oxygen workstation (0.1% O₂). One hour layer, the culture medium was recovered,trypsin was added, and the cells were recovered. The recovered cellswere disrupted by sonic treatment and the compound was extracted withacetonitrile, following which analysis by LC/MS of the amount ofprednisolone released was carried out under the following conditions.

Column used: TSKgel ODS-100Z (particle size, 3 μm; 2 mm×75 mm) column

Measurement wavelength: 250 nm

Eluent A: 0.1 M ammonium acetate

Eluent B: acetonitrile

Flow rate: 0.2 mL/min

Gradient: 40:60 (Eluent A:Eluent B)→10:90 (20 minutes)

By carrying out this measurement, the amount of compound released in alow-oxygen environment was compared with the amount released in a normaloxygen environment. The HPLC results are shown in FIG. 8. It is apparentfrom the diagram than much prednisolone is released in a low-oxygenenvironment.

INDUSTRIAL APPLICABILITY

This invention makes it possible to provide prodrugs which reduce theside effects of the parent compound in a normal oxygen concentrationenvironment, but exhibit the activity inherent to the parent compound ina low-oxygen environment. This invention can be utilized in, forexample, the pharmaceutical industry which provides therapeuticallyactive organic compounds of reduced toxicity.

The invention claimed is:
 1. A compound represented by general formula(I),

wherein Z is of formula (a)

or of formula (b)—O—R3  (b), R1 being a residue of an amino group-bearing therapeuticallyactive organic compound after removal of the amino group therefrom andR2 being a hydrogen atom, or R1 and R2, taken together with theadjoining nitrogen atom, being a residue of a therapeutically activeorganic compound having a cyclic amino group; and R3 being a residue ofa hydroxyl group-bearing therapeutically active organic compound afterremoval of the hydroxyl group therefrom, wherein the amino group-bearingtherapeutically active organic compound is selected from the groupconsisting of doxorubicin and gemcitabine; the therapeutically activeorganic compound having a cyclic amino group is represented by5-fluorouracil; the hydroxyl group-bearing therapeutically activeorganic compound is selected from the group consisting of doxorubicinand gemcitabine; and the moiety corresponding to Z of general formula(I) is cleaved in a reducing environment, or a pharmaceuticallyacceptable salt thereof.
 2. The compound according to claim 1 which hasa formula as follows:


3. The compound according to claim 1 which has a formula as follows:


4. The compound according to claim 1 which has one of the following twoformulae: